The Smi-miR858a-SmMYB module regulates tanshinone and phenolic acid biosynthesis in Salvia miltiorrhiza

Abstract Tanshinones and phenolic acids are two major classes of bioactive compounds in Salvia miltiorrhiza. Revealing the regulatory mechanism of their biosynthesis is crucial for quality improvement of S. miltiorrhiza medicinal materials. Here we demonstrated that Smi-miR858a–Smi-miR858c, a miRNA family previously known to regulate flavonoid biosynthesis, also played critical regulatory roles in tanshinone and phenolic acid biosynthesis in S. miltiorrhiza. Overexpression of Smi-miR858a in S. miltiorrhiza plants caused significant growth retardation and tanshinone and phenolic acid reduction. Computational prediction and degradome and RNA-seq analyses revealed that Smi-miR858a could directly cleave the transcripts of SmMYB6, SmMYB97, SmMYB111, and SmMYB112. Yeast one-hybrid and transient transcriptional activity assays showed that Smi-miR858a-regulated SmMYBs, such as SmMYB6 and SmMYB112, could activate the expression of SmPAL1 and SmTAT1 involved in phenolic acid biosynthesis and SmCPS1 and SmKSL1 associated with tanshinone biosynthesis. In addition to directly activating the genes involved in bioactive compound biosynthesis pathways, SmMYB6, SmMYB97, and SmMYB112 could also activate SmAOC2, SmAOS4, and SmJMT2 involved in the biosynthesis of methyl jasmonate, a significant elicitor of plant secondary metabolism. The results suggest the existence of dual signaling pathways for the regulation of Smi-miR858a in bioactive compound biosynthesis in S. miltiorrhiza.

Although the involvement of miR858 in regulating f lavonoid biosynthesis has been revealed in several plants, its regulatory role for other secondary metabolites remains largely unknown.In this study, we identified MIR858 in the S. miltiorrhiza genome and comprehensively analyzed its regulatory role in bioactive compound biosynthesis through a combination of computational prediction, genetic transformation, degradome analysis, RNAsequencing (RNA-seq) analysis, ultra-high performance liquid chromatography (UPLC) detection, yeast one-hybrid assay (Y1H), and transient transcriptional activity assay (TTA).The results suggest that, in addition to f lavonoid biosynthesis, S. miltiorrhiza miR858 also plays important roles in regulating phenolic acid and tanshinone biosynthesis through direct cleavage of SmMYB6, SmMYB97, SmMYB111, and SmMYB112 transcripts.It subsequently affects the expression of genes involved in bioactive compound biosynthesis pathways.In addition, SmMYB6, SmMYB97, and SmMYB112 can activate SmAOC2, SmAOS4, and SmJMT2 involved in the MeJA biosynthetic pathway, which may subsequently stimulate the biosynthesis of bioactive compounds.The results reveal a novel function of miR858 and provide important information for improving the quality of medicinal materials.

S. miltiorrhiza
To identify all candidate MIR858 genes in S. miltiorrhiza, we first carried out BLASTn analysis of known miR858 in miRBase (release 22.1) against the S. miltiorrhiza sRNA database [37][38][39][40][41][42].This resulted in the identification of three mature Smi-miR858 sequences, termed Smi-miR858a, Smi-miR858b, and Smi-miR858c (Fig. 1A).Among them, Smi-miR858a and Smi-miR858c differ in the last nucleotide of the 3 end, whereas Smi-miR858b has three and two nucleotide differences from Smi-miR858a and Smi-miR858c, respectively (Fig. 1A).We then mapped the three mature Smi-miR858 sequences to the genome of S. miltiorrhiza line 99-3 and predicted secondary structures for the genome sequence surrounding Smi-miR858s using RNA folding (Fig. 1B, Supplementary Data Figs S1-S3) [43,44].The predicted secondary structures were manually checked based on the criteria proposed by Meyers et al. [45].As a result, a total of three S. miltiorrhiza MIR858 precursors (Smi-MIR858a-Smi-MIR858c) were identified.Among them, Smi-MIR858a and Smi-MIR858c are located on scaffold 2321 with an interval of 2856 bp.Smi-MIR858b is located on scaffold 7869 (Fig. 1C).This suggests that the three Smi-MIR858 genes identified by computational prediction are authentic and expressed.To further characterize the function of Smi-miR858, the expression level of Smi-miR858s was analyzed using sRNA data identified from five S. miltiorrhiza root small RNA libraries.As shown in Supplementary Data Fig.S4, Smi-miR858a was expressed at the highest levels in all five root tissues,

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Smi-miR858c was expressed at moderate levels, and Smi-miR858b was expressed at the lowest level.

Smi-miR858s targeted R2R3-MYB transcription factor genes in S. miltiorrhiza
Plant miRNAs can guide RNA-induced silencing complexes to target transcripts and then silence the targets mainly by direct cleavage at a site that corresponds to the 10th nucleotide from the miRNA 5 end [25,46].The existence of perfect or nearperfect complementarity between miRNAs and their targets is critical in the process of target reorganization and subsequent cleavage.Thus, computational analysis of complementarity is an effective approach in predicting plant miRNA targets [47].We first predicted the targets of Smi-miR858a on the online web servers TAPIR and psRNAtarget using the default parameters [48,49].This resulted in the prediction of a total of 11 targets (Fig. 2A, Supplementary Data Fig.S5, Supplementary Data Table S1), of which SmMYB111 was targeted by three Smi-miR858s.SmMYB6, SmMYB88, SmMYB97, and SMil_00003526 were targeted by Smi-miR858a and Smi-miR858c.The other six were targeted by either Smi-miR858a or Smi-miR858b (Supplementary Data Fig.S5, Supplementary Data Table S1).The Smi-miR858-complementary site of these R2R3-SmMYB transcripts was located in a region encoding the conserved imperfect repeat 3 (R3) [12].
Next, we aligned the degradome data of S. miltiorrhiza to the putative SmMYB targets.Significant accumulation of degraded fragments corresponding to the predicted cleavage sites were found for SmMYB6, SmMYB97, SmMYB111, and SMil_00003526 (Fig. 2B).This suggests that the four SmMYBs are authentic targets of Smi-miR858s.Among them, SmMYB97 has been previously shown to promote tanshinone and phenolic acid accumulation by activating the promoters of SmCPS1, SmKSL1, SmPAL1, and SmTAT1 [18].SmMYB111 positively regulates phenolic acid biosynthesis by forming a transcription complex with SmTTG1 and SmbHLH51 [17].SmMYB6 is a functionally unknown SmMYB identified in our previous studies [12].SMil_00003526 is a novel SmMYB that has not been reported before.Here we named it SmMYB112.Phylogenetic analysis and multiple sequence alignment of SmMYB6, SmMYB97, SmMYB111, SmMYB112, and various R2R3-MYBs identified in Arabidopsis thaliana, Zea mays, Malus domestica, and Vitis vinifera showed that SmMYB6, SmMYB97, and SmMYB111 were R2R3-MYBs belonging to subgroup 7, whereas SmMYB112 was clustered with R2R3-MYBs in subgroup 6 (Supplementary Data Figs S6-S8, Supplementary Data Table S4).Except for SmMYB6, SmMYB97, SmMYB111, and SmMYB112, another seven predicted SmMYB targets could not be validated through degradome analysis.This indicates that these predicted targets could not be authentic targets of Smi-miR858s.The other possibility could be that the degradome data were not sufficient to validate these predicted targets.

Overexpression of Smi-miR858a in S. miltiorrhiza plants repressed SmMYB targets and caused growth retardation
Since Smi-miR858a had the greatest number of putative targets among the identified three Smi-miR858s, could regulate the four validated SmMYB targets, and showed the highest expression in tissue analysis (Supplementary Data Figs S4 and S5, Supplementary Data Table S1), it was selected for transgenic analysis.To overexpress Smi-miR858a in S. miltiorrhiza, an artificial microRNA (amiRNA) vector, termed pHPT-858, was designed and constructed as described previously [50].Ptc-MIR408 precursor identified from Populus trichocarpa was used as the backbone [47].Smi-miR858a and Smi-miR858a * sequences were incorporated into the backbone to replace ptc-miR408 and ptc-miR408 * sequences, respectively.The constructed pHPT-858 was introduced into S. miltiorrhiza line 99-3 by Agrobacterium tumefaciens-mediated transformation as described before [51].A total of 19 transgenic lines were obtained.
Analysis of the Smi-miR858a level in transgenic lines using an miRNA-specific stem-loop RT-qPCR method [28] showed that the content of Smi-miR858a was increased dramatically (Fig. 3A).The expression level of SmMYB targets in transgenics and wild-type plants was comparatively analyzed using the RT-qPCR method.This showed that all four SmMYB genes were significantly downregulated in Smi-miR858a overexpression lines (Fig. 3B-E), and provides solid evidence for Smi-miR858a-mediated regulation of SmMYB6, SmMYB97, SmMYB111, and SmMYB112 in S. miltiorrhiza.In accompaniment with the increase in Smi-miR858a and the downregulation of SmMYB targets, the transgenic plants showed significant growth retardation characterized by smaller leaves and roots and fewer leaves and roots (Fig. 3F).Fresh weight was decreased from 15.50 g in the wild type to 8.08 g in transgenic lines (Fig. 3G).

Overexpression of Smi-miR858a significantly reduced tanshinone and phenolic acid production
It has been shown that miR858 regulates f lavonoid biosynthesis in some plants [28,30,32,35,36].However, there is no information about its regulatory role for other secondary metabolites.In order to gain insight into the precise role of Smi-miR858a in regulating secondary metabolism, we analyzed the content of phenolic acids and tanshinones in roots [1].Dried roots of 3-month-old wild-type plants and artificial Smi-MIR858a transgenics were collected and analyzed using UPLC.The results showed that, compared with the wild-type plants, the average contents of RA and Sal B in Smi-miR858a transgenics were reduced by 73.2 and 87.2%, respectively (Fig. 4A and B).Analysis of the four major tanshinones, CT, DT-I, TAII, and TAI, showed that their average contents in roots of Smi-MIR858a transgenics were decreased by 65.9, 58.5, 53.7, and 70.4%, respectively (Fig. 4C-F).The results indicate that Smi-miR858a negatively regulates phenolic acid and tanshinone biosynthesis in S. miltiorrhiza.

Effects of Smi-miR858a on global gene expression
To further clarify the biological role of miR858 in S. miltiorrhiza, RNA-seq analysis was performed to detect differentially expressed genes (DEGs) between wild-type and miR858 overexpression lines.The analysis was carried out for roots from a 3-month-old wild-type line and three transgenic lines.Three replicates for each line were performed.The average value of FPKM (fragments per kilobase of transcript per million fragments mapped) was used for comparison.Significantly upand downregulated genes (|log2(foldchange)| > 2 and P < 0.05) were screened.This resulted in the identification of 845 DEGs, of which 305 were upregulated and 540 downregulated (Supplementary Data Table S3).KEGG analysis showed that DEGs involved in 'phenylpropanoid biosynthesis', 'phenylalanine, tyrosine and tryptophan biosynthesis', 'alpha-linolenic acid metabolism', 'terpenoid backbone biosynthesis', 'f lavonoid biosynthesis', and 'stilbenoid, diarylheptanoid and gingerol biosynthesis' were significantly enriched (Fig. 5).The regulatory role of miR858 in f lavonoid biosynthesis has been previously found in various plants, such as Arabidopsis, persimmon, apple, and kiwifruit [28,30,32,35,36].The enrichment of DEGs in the f lavonoid biosynthesis pathway suggests that Smi-miR858a plays a conserved function in regulating f lavonoid biosynthesis in S. miltiorrhiza.The enrichment of DEGs in the major bioactive compound biosynthesis pathways, such as the terpenoid backbone biosynthesis pathway for tanshinone production and the phenylpropanoid biosynthesis pathway related to phenolic acid production, is consistent with the results from phenolic acid and tanshinone content determination (Fig. 4).In addition, the α-linolenic acid metabolism pathway is associated with the production of MeJA [21,24].The enrichment of DEGs in this pathway provides another layer of evidence for elucidating the role of Smi-miR858 in regulating bioactive compound biosynthesis.

Smi-miR858a targeted SmMYBs to regulate the expression of downstream genes
Through computational prediction and degradome analysis, we found that SmMYB6, SmMYB97, SmMYB111, and SmMYB112 were the targets of Smi-miR858s (Fig. 2).Consistently, overexpression of Smi-miR858a caused significant downregulation of the four SmMYBs (Fig. 3).Among them, SmMYB97 activates tanshinone and phenolic acid biosynthesis [18].SmMYB111 is a member of the transcription complex SmTTG1-SmMYB111-SmbHLH51.It positively regulates the expression of genes involved in phenolic acid biosynthesis [17].The functions of SmMYB6 and SmMYB112 remain to be elucidated.
Next, we asked whether SmMYB6 and SmMYB112 could activate the transcription of SmCPS1 SmKSL1, SmPAL1, and SmTAT1.In order to address this question, a transient transcriptional activity [29] assay was conducted.The promoter regions of SmCPS1 SmKSL1, SmPAL1, and SmTAT1 used for Y1H were fused with the LUC reporter gene.The vectors were then co-transformed into  tobacco leaves with 35S:GFP, 35S:Smi-miR858a, 35S:SmMYB6, or 35S:SmMYB112.Analysis of the luminescence intensity showed that SmMYB6 and SmMYB112 could significantly increase the luminescence intensity (Fig. 8B-F).This indicates that both SmMYB6 and SmMYB112 can activate the expression of SmKSL1, SmCPS1, SmPAL1, and SmTAT1.In addition, co-transformation of 35S:Smi-MIR858a and 35S:SmMYB6 or 35S:Smi-MIR858a and 35S:SmMYB112 with the reporters into tobacco leaves resulted in significant reduction of f luorescence intensity in comparison with those without 35S:Smi-miR858a.It further confirms that Smi-miR858a negatively regulated the expression of SmMYB6 and SmMYB112.
Smi-miR858a targeted SmMYBs to regulate methyl jasmonate biosynthesis genes KEGG analysis showed that DEGs involved in the α-linolenic acid metabolism pathway were enriched (Fig. 5).Further analysis of genes involved in the pathway showed that SmAOS1, SmAOS4, SmAOS6, SmAOS7, SmAOC1, SmAOC2, and SmJMT2 were downregulated in Smi-miR858a overexpression plants in comparison with their expression in wild-type plants (Fig. 9A).To reveal the regulatory role of SmMYB6/97/111/112 in MeJA biosynthesis, Y1H and TTA experiments were performed.Since the expression of SmAOC2, SmAOS4, and SmJMT2 showed the most significant downregulation, their promoter regions were selected for further analysis.As shown in Fig. 9B, all three promoters contain the MYB-responsive elements.
For the TTA assay, the same promoter regions of SmAOC2, SmAOS4, and SmJMT2 used for Y1H were fused with the reporter gene and co-transformed into tobacco leaves with 35S:GFP, 35S:SmMYB6, 35S:SmMYB97, 35S:SmMYB111, 35S:SmMYB112, or 35S:Smi-miR858a.Luminescence intensity measurement showed that the expression of SmAOC2 could be significantly activated by SmMYB6 and SmMYB97.The expression of SmAOS4 could be significantly activated by SmMYB97 and SmMYB112.The expression of SmJMT2 could be activated by SmMYB6 and SmMYB97, although the degree of activation was relatively low in comparison with the activation of SmMYB6 and SmMYB97 on SmAOC2 and SmMYB97 and SmMYB112 on SmAOS4 (Fig. 9D).This also showed that the activation activities were inhibited by Smi-miR858a.The results are consistent with those from Y1H (Fig. 9C), suggesting regulatory role of Smi-miR858a in MeJA biosynthesis through targeting SmMYB transcripts.

MiR858 is a key regulator of bioactive compound biosynthesis in plants
MYBs, characterized by the deeply conserved MYB domain, are composed of the largest transcription factor family in plants.Members of the family play significant regulatory roles in the biosynthesis of bioactive compounds [1,25].Several miRNAs, including miR159, miR319, miR828, and miR858, are involved in the post-transcriptional regulation of plant MYB genes [12].Among them, miR858 has the greatest number of MYB targets, since its target site is located in the deeply conserved 3 end of the R3 repeats of MYB genes [12].Previous studies have shown that several miR858-targeted MYB genes are involved in f lavonoid biosynthesis [28,30,32,35,36].Through cleaving the transcripts of these MYB genes, miR858 could regulate the biosynthesis Figure 8. Binding of SmMYB6 and SmMYB112 to tanshinone and phenolic acid biosynthesis-related genes.A Binding of SmMYB6 and SmMYB112 to SmPAL1, SmTAT1, SmCPS1, and SmKSL1 promoters in yeasts.p53HIS2/pGADT7-p53 vectors were used as positive control.p53HIS2/pGADT7-SmMYB6 and p53HIS2/pGADT7-SmMYB112 were used as negative controls.B Schemes of the reporter and effector constructs used in transient transcriptional activity.TBG, tanshinone biosynthesis genes.PBG, phenolic acid biosynthesis genes.C-F Transcriptional activity of SmMYB6 and SmMYB112 on the promoters of SmPAL1, SmTAT1, SmCPS1, and SmKSL1 in tobacco leaves.Bars are standard deviations of three biological replicates.Lowercase letters indicate a significant difference determined by one-way ANOVA and post hoc Tukey's test (P = 0.05).
Salvia miltiorrhiza Bunge is a perennial plant in the Salvia genus of the Lamiaceae family [1].The main bioactive compounds, phenolic acids, and tanshinones, have been clinically used for management of vascular diseases and various other diseases [2,3].In addition, S. miltiorrhiza also produces other bioactive compounds, such as anthocyanidins, f lavonoids, monoterpenes, sesquiterpenes, proanthocyanidins, triterpenes, and so on [1].Overexpression of Smi-miR858a caused significant downregulation of f lavonoid biosynthesis enzyme genes, such as SmANS, SmCHI1, and SmF3 H2.This suggests that the regulatory role of miR858 in f lavonoid biosynthesis is conserved among S. miltiorrhiza and other eudicotyledon species.In addition to f lavonoids, overexpression of Smi-miR858a also caused significant decreases in tanshinone and phenolic acid contents.The results reveal novel functions of miR858 and suggest that Smi-miR858a is a negative regulator of phenolic acid, tanshinones, and f lavonoids in S. miltiorrhiza.Considering the significance of MYBs in secondary metabolism and the conservation of miR858 in plants, it is very likely that the regulatory role of miR858 in the biosynthesis of phenolic acids and terpenoids in S. miltiorrhiza is also conserved in other plant species, although this hypothesis remains to be tested.In addition, Smi-miR858aoverexpressing transgenic plants showed significant growth retardation characterized by smaller leaves and roots and fewer leaves and roots (Fig. 3F).This indicates that Smi-miR858a also plays important roles in plant growth and development.

MiR858 regulated bioactive compound biosynthesis through dual signaling pathways
Through computational analysis, we predicted a total of 11 SmMYBs to be targets of three Smi-miR858s.Among them, SmMYB6, SmMYB97, SmMYB111, and SmMYB112 were validated through degradome analysis and Smi-miR858a overexpression.Previous studies suggest that SmMYB97 acts as a positive regulator of tanshinone and phenolic acid biosynthesis [18].

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SmMYB111 activates phenolic acid biosynthesis through forming a transcription complex with SmbHLH51 and SmTTG1 [17].Here, we analyzed the function of SmMYB6 and SmMYB112 using Y1H and TTA assays.We found that both of them could activate the expression of SmKSL1, SmCPS1, SmPAL1, and SmTAT1, of which SmKSL1 and SmCPS1 are involved in tanshinone biosynthesis, whereas SmPAL1 and SmTAT1 are involved in phenolic biosynthesis.Since overexpression of Smi-miR858a significantly not only downregulated the four enzyme genes mentioned above but also various other genes related to f lavonoid tanshinone and phenolic acid biosynthesis (Figs 6 and 7, Supplementary Data Fig, S9), we could not exclude the possibility that some of these enzyme genes were also regulated by the SmMYB targets of Smi-miR858a.
In addition to binding the promoters of bioactive compound biosynthesis pathway genes, SmMYB6, SmMYB97, and SmMYB112 could also activate genes involved in the MeJA biosynthesis pathway through binding to the promoters of these genes (Fig. 9).Among them, SmMYB6 could activate the expression of SmAOC2 and SmJMT2.SmMYB97 could activate the expression of SmAOC2, SmAOS4, and SmJMT2.SmMYB112 could activate the expression of SmAOS4.Since overexpression of MeJA biosynthesis-related genes and external application of MeJA could significantly enhance phenolic acid and tanshinone production in S. miltiorrhiza [21][22][23][24], the upregulation of SmAOC2, SmAOS4, and SmJMT2 by Smi-miR858a-targeted SmMYB6, SmMYB97, and SmMYB112 suggests another layer of the signaling pathway for the regulation of Smi-miR858a in bioactive compound biosynthesis.
Recently, Zheng et al. overexpressed Smi-miR396b in hairy roots of S. miltiorrhiza [59].They found that Smi-miR396b downregulated salvianolic acid biosynthesis and upregulated tanshinone production through targeting growth-regulating factor genes (SmGRFs), histone deacetylase gene (SmHDT1), and SmMYB37/4 genes.In addition, Zou et al. constructed an amiRNAmediated miR408-suppressed expression vector to inhibit the expression of Smi-miR408 in S. miltiorrhiza plants [60].The results showed that the content of phenolic acids increased in the transgenic lines.Further analysis showed that the regulatory role of Smi-miR408 in phenolic acid biosynthesis was mediated by targeting the transcripts of laccase gene (SmLAC3) gene for cleavage [60].In this study, we found that Smi-miR858a played significant regulatory role in the biosynthesis of phenolic acids, tanshinones, and f lavonoids through targeting a subset of SmMYB genes.This further confirms the significance of miRNA-mediated post-transcriptional regulation in bioactive compound production in S. miltiorrhiza.
Because of their vital regulatory role in secondary metabolite biosynthesis, miRNAs of medicinal plants have become a bright research field [25].But information on the function and regulatory network of medicinal plant miRNAs is very limited.Greater efforts are needed to authenticate medicinal plant miRNA candidates and uncover their functions through genetic transformation.From this point of view, elucidating the regulatory role of Smi-miR858 in bioactive compounds is meaningful.

Plant materials and growth conditions
Sterile plantlets of S. miltiorrhiza (line 99-3) were grown on 1/2MS agar medium at 25 • C for 40 days under a photoperiod of 16 h light/8 h dark.Leaf discs from the sterile plantlets were used for Agrobacterium-mediated transformation as described previously [51].For transient transcriptional activity assay [29], Nicotiana benthamiana seeds were sown in soil and grown at 25 • C for 1 month with the photoperiod of 16 h light/8 h dark in a greenhouse.

Plasmid construction and plant transformation
Artificial microRNA 858a (amiR858a) was designed as previously reported [50].The pBI-MIR408 binary vector was used as the template.Three pairs of primers were designed (Supplementary Data Table S2) and used in different combinations to conduct PCR amplification.The three prime combinations were 35sP/4P, 3P/2P, and 1P/NosP, respectively.The amplified three fragments contained overlapping sequences of Smi-miR858a and Smi-miR858a * .Overlapping PCR using the 35SP/NosP primer set generated a new fragment containing the Smi-miR858a and Smi-miR858a * sequences.The fragment was digested with SacI and BamHI, and then cloned into pMD18-T vector.After sequence verification, the fragment was inserted into pGPTV-HPT to generate the pHPT-858 overexpression vector.The vector was transformed into A. tumefaciens GV3101.Agrobacterium-mediated transformation of S. miltiorrhiza was conducted as previously described [51].

Figure 1 .
Figure 1.Analysis of Smi-miR858a/b/c in S. miltiorrhiza.A Sequences of Smi-miR858a, Smi-mi858b, and Smi-mi858c.Letters in red indicate different nucleotides among the three miRNAs.B Partial hairpin structures of Smi-MIR858a, Smi-MIR858b, and Smi-MIR858c.Smi-miR858a, Smi-miR858b, and Smi-miR858c sequences are indicated in red.Numbers indicate nucleotides numbered from the 5 end in the corresponding precursors.C Schemes for the position of Smi-MIR858a, Smi-MIR858b, and Smi-MIR858c precursors in the scaffolds.

Figure 2 .
Figure 2. Analysis of Smi-miR858 targets.A Binding sites of Smi-miR858a in SmMYB6, SmMYB97, SmMYB111, and SmMYB112.Arrows indicate the 5 termini of miRNA-guided cleavage products.B Degradome analysis of Smi-miR858a-mediated cleavage of SmMYB6, SmMYB97, SmMYB111, and SmMYB112.The X-axis shows the nucleotide position of the gene and the Y-axis shows the number of reads obtained by degradome sequencing.Black lines represent degradome fragments matched to the genes.Arrows indicate the products cleaved by Smi-miR858a.

Figure 3 .
Figure 3. Analysis of Smi-miR858a overexpression in S. miltiorrhiza.A Relative expression level of Smi-miR858a in transgenic and wild-type lines.Bars represent standard deviations of the mean from three biological replicates ( * * P < 0.01, Student's t-test).B Relative expression level of SmMYB6 in transgenic and wild-type lines.C Relative expression level of SmMYB97 in transgenic and wild-type lines.D Relative expression level of SmMYB111 in transgenic and wild-type lines.E Relative expression level of SmMYB112 in transgenic and wild-type lines.Bars are standard deviations of three biological replicates ( * * P < 0.01, Student's t-test).F Phenotype of Smi-miR858a overexpression and wild-type line.Scale bar = 5 cm.G Comparison of fresh weight of wild-type and transgenic plants.Values are standard deviations determined by Student's t-test (n = 15, * * P < 0.01).

Figure 4 .
Figure 4. Analysis of phenolic acid and tanshinone contents in transgenic and wild-type lines.Contents of RA (A), Sal B (B), CT (C), DT-I (D), TAII (E) and TAI (F) in transgenic and wild-type roots.Bars represent standard deviations of the mean for three biological replicates determined by Student's t-test ( * * P < 0.01).

Figure 5 .
Figure 5. KEGG analysis of DEGs in transgenic and wild-type lines.The top 21 most statistically significant terms of KEGG pathways.The X-axis represents GeneRatio and the Y-axis represents KEGG pathways.The size of circle represents gene number.Different color of circles represents adjusted P value.

Figure 6 .
Figure 6.RNA-seq analysis of genes involved in the phenolic acid biosynthesis pathway in transgenic and wild-type lines.The average FPKM of three biological replicates was used for comparison.

Figure 7 .
Figure7.RNA-seq analysis of genes involved in tanshinone biosynthesis pathway in transgenic and wild-type lines.The average FPKM of three biological replicates was used for comparison.

Figure 9 .
Figure 9. Binding of SmMYBs to MeJA biosynthesis-related genes.A RNA-seq analysis of genes involved in MeJA biosynthesis pathway in transgenic and wild-type lines.The average FPKM of three biological replicates was used for comparison.B Distribution of MYB-binding sites in SmAOC2, SmAOS4, and SmJMT2 promoters.MBS1, CAACTG; MRS, CCGTTG; MBS2, CAACCA; MBS3, TAACCA; MRE, AACCTAA.C Binding analysis of SmMYB6, SmMYB97, SmMYB111, and SmMYB112 to SmAOC2, SmAOS4, and SmJMT2 promoters in yeasts.p53HIS2 and pGADT7-p53 were used as positive controls.p53HIS2 and pGADT7-SmMYBs were used as negative controls.D Transcriptional activity of SmMYB6, SmMYB97, SmMYB111, and SmMYB112 on the promoters of SmAOC2, SmAOS4, and SmJMT2 in tobacco leaves.Bars are standard deviations of three biological replicates.Lowercase letters indicate significant difference determined by one-way ANOVA and post hoc Tukey's test (P = 0.05).